The primary control point for transepithelial electrolyte transport is C1- secretion across the apical membrane. Several types of external receptors, coupled to a variety of effector systems (CAMP, Ca2+, CGMP, PKC) mediate C1- secretion. Cystic fibrosis (CF) is characterized by abnormal C1- secretion in epithelial cells, with some regulatory pathways affected and others preserved. CAMP-dependent C1- secretion, induced by CAMP kinase-mediated phosphorylation of cystic fibrosis transmembrane conductance regulator (CFTR), is affected in CF. Ca2+-dependent C1- secretion, induced by CaM kinase-mediated phosphorylation of an independent C1- channel, is preserved in CF. Delineation of C1-transport pathways which are preserved in CF presents potential targets for therapeutic intervention. The overall goal of this proposal is, therefore, to investigate other regulatory pathways of C1- secretion that may ultimately serve to bypass the CF defect. The techniques employed will be patch clamp recording, supplemented by spectrofluorimetry and I125 efflux assays, of normal and CF-derived airway and intestinal epithelial cell lines. The specific aims include the investigation of' i) CGMP-dependent C1- secretion: to characterize currents mediated by heat stable enterotoxin (ST) in intestinal cells and by CPT-CGMP in airway cells; by use of coexpression of the ST receptor and CFTR in nonepithelial cells, to determine whether the pathway is CF-affected. ii) PKC-dependent C1- secretion: to determine by immunoblot the specific PKC isoforms expressed in epithelial cells; to characterize PKC-dependent regulation of C1- transport; to determine status in CF. iii) purinoreceptor pathway of C1- secretion; to determine the predominant receptor subtype by which adenosine and ATP analogues stimulate secretion; to investigate the responsible effector system; to determine status in CF.